The long-term objective of this project is to understand the role of protein kinase R (PKR) in the regulation of signal transduction pathways activated by interferons (IFNs), cytokines, and the pathogen associated molecular patterns (PAMPS) signaling via Toll like receptors (TLRs). PKR regulates signaling in the NF-tcB pathway by different ligands including dsRNA, tumor necrosis factor (TNF) and IFN-y via its interaction with ItcB kinase. The engagement of the Type I and type IIIFN receptors can also lead to PKR regulation of cytosolic cPLA2 and Statl serine phosphorylation. In addition, Stat3 tyrosine and serine phosphorylation can be regulated via a PKR-dependent pathway. PAMP engagement of TLRs also activates PKR-dependent regulation of the production of pro-inflammatory cytokines via a mitogen activated protein kinase (MAPK) pathway where PKR interacts with MAP kinase kinase 6 (MKK6). Our discoveries that dsRNA can regulate tumor suppressor p53 stability and that dsRNA and CpG oligodeoxynucleotides (CpG ODN) can synergize in pro-inflammatory cytokine production raises questions about a role for PKR. We propose to provide a better understanding of the molecular determinants that regulate these pathways. More specificallywe will focus on the role of PKR as a key signaling molecule that can be invoked by IFNs, cytokines, and PAMPs to signal different transcription factors regulating gene expression. We will achieve this by the following specific aims. We will establish the molecular determinants that define PKR as a key regulator of MAPK signaling pathways. We will investigate the mechanisms of p53 instability induced by dsRNA and characterize the signaling.pathway by which this is achieved. We will define the mechanisms of synergy between dsRNA and CpG ODN by investigating the role of type I IFNs, dsRNA signaling pathways, and components of the TLR signaling pathway required to mediate the synergistic response. We will also determine whether the synergy results in enhanced tumor immunity in vivo and establish the relative contribution of the dsRNA-activated pathways. These studies have the potential to provide insight into the regulation of innate immunity and its exploitation in cancer therapy.